Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as “features”) are positioned at respective locations (“addresses”) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample. Biopolymer arrays can be fabricated by depositing previously obtained biopolymers (such as from synthesis or natural sources) onto a substrate, or by in situ synthesis methods. Methods of depositing obtained biopolymers include dispensing droplets to a substrate from dispensers such as pin or capillaries (such as described in U.S. Pat. No. 5,807,522) or such as pulse jets (such as a piezoelectric inkjet head, as described in PCT publications WO 95/25116 and WO 98/41531, and elsewhere). The substrate is coated with a suitable linking layer prior to deposition, such as with polylysine or other suitable coatings as described, for example, in U.S. Pat. No. 6,077,674 and the references cited therein.
For in situ fabrication methods, multiple different reagent droplets are deposited from drop dispensers at a given target location in order to form the final feature (hence a probe of the feature is synthesized on the array substrate). The in situ fabrication methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, and described in WO 98/41531 and the references cited therein for polynucleotides. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides from nucleoside reagents on a support by means of known chemistry. This iterative sequence is as follows: (a) coupling a selected nucleoside through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e. the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (“deprotection”) from the now substrate bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, in a known manner. As can be seen, in situ fabrication involves multiple cycles, whereas the deposition of previously obtained biopolymers is generally one cycle (that is, only one occurrence of probes occurs at each feature).
The foregoing chemistry of the synthesis of polynucleotides is described in detail, for example, in Caruthers, Science 230: 281–285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323–356; Hunkapillar et al., Nature 310: 105–110, 1984; and in “Synthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivatives”, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and elsewhere. Suitable linking layers on the substrate include those as described in U.S. Pat. Nos. 6,235,488 and 6,258,454 and the references cited therein.
Further details of fabricating biopolymer arrays by depositing either previously obtained biopolymers or by the in situ method are disclosed in U.S. Pat. No. 6,242,266, U.S. Pat. No. 6,232,072, U.S. Pat. No. 6,180,351, and U.S. Pat. No. 6,171,797.
In array fabrication, the quantities of DNA available for the array are usually very small and expensive. Sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced features. It is important in such arrays that features actually be present, that they are put down accurately in the desired pattern, are of the correct size, and that the DNA is uniformly coated within the feature. Normally, in an automated apparatus the features are deposited according to a target array pattern. A target drive pattern is created from the target array pattern, which target drive pattern contains the instructions for driving the various components so as to provide the probes on the substrate in the target array pattern. The target drive pattern is created on the assumption that all components of the deposition apparatus are in their expected or normal (“nominal”) positions and operating according to nominal parameters.
However, components in an array deposition apparatus each are subject to variances in its parameters within, or sometimes even outside of, normal tolerances for such component. For example, a dispensing head used to dispense fluid droplets to form the array, may have jets which vary slightly in the size of the droplets dispensed, the orientation of the jets with respect to one another, or the orientation of the head itself in the apparatus may be slightly off from a nominal position. While such variances can be reduced by constructing a dispensing apparatus with components of higher tolerance (that is, less variation), this can increase cost. Furthermore, while a given set of parameters may exist during manufacture of a given batch of arrays, these parameters may change over time, for example due to thermal expansion of a component. These effects result in use of the target drive pattern not producing the target array on the substrate. That is, there is a discrepancy between the target array pattern and the actual array pattern deposited. Such discrepancy may include mislocation of features, or features not being of the correct size. These discrepancies can occur in each cycle of the in situ process, or during deposition of presynthesized polynucleotides.
Errors of the foregoing type can be monitored and corrected to extent by detecting the positions of deposited drops and calculating their positions based on encoder information which provides feedback on component positions (such as the position of the head). However, the present invention realizes that while such a procedure can be highly useful it can have limitations. For example, encoder errors may vary over time. Also, these or other drop deposition errors may be non-linear (that is their magnitude may vary with the position of a drop deposition head relative to the substrate). Such further minor positioning errors may not be significant in typical sized substrates onto which multiple arrays are fabricated. However, the present invention further realizes that to increase manufacturing throughput substrate size should be increased to increase the number of arrays that can be simultaneously fabricated on the substrate, and that in such event errors of the foregoing type become more significant (primarily due to the longer distance of travel of the deposition head relative to the substrate).
It would be useful then, to provide a means by which arrays can be fabricated with an actual array pattern which is very close to the target array pattern. It would also be useful if such means was relatively reliable and easy to implement.